Disease-sites, like cancer cells, (over)express “finger-print” biomolecules, such as antigens or receptors, which may serve as recognition sites for a wide range of circulating vectors, such as antibodies, peptide-hormones, growth factors etc. One approach to diagnose and identify disease-sites is to exploit such interaction and to use a suitably modified vector, such as a peptide analog labeled with a diagnostic radionuclide, which will specifically accumulate on the disease-site(s) after administration to the patient. The tumor and metastases are then localized by imaging the site(s) where the radioactive decay occurs using an external imaging device.
A similar rationale is followed in targeted therapy, whereby the vector will again serve as the vehicle which will deliver a cytotoxic load, such as a therapeutic radiometal, specifically to the disease-sites, e.g. the tumor and metastases. The therapeutic radiolabel will then decay on the disease site, releasing particle radiation to kill or to reduce the growth of the tumor.
The efficacy of targeted diagnosis and treatment is often compromised by degradation of the administered site-specific drug by endogenous enzymes. Enzymatic breakdown may occur in the blood stream immediately after entry into circulation and until the drug reaches the target. Metabolic attack may be operated during transit by enzymes circulating in the blood solute, but most importantly, by enzymes anchored on the membrane of blood cells, vasculature walls and several tissues of the human body (liver, kidneys and gastrointestinal tract) including tumor tissue. These enzymes will greatly affect drug delivery. Furthermore, the micromilieu around the target, as for example the peritumoral environment (stroma cells, local (neo)vasculature and extracellular matrix), is another potential degradation site for diagnostic and therapeutic drugs, likely to affect not only accumulation but also retention at the target.
It has long been established, that the action of many endogenous substances, such as peptide-hormones, is regulated by enzymes, both in normal conditions and during cancer onset and propagation. Thus, an “intimate” relationship seems to exist for example between G protein coupled receptors (GPCRs), their peptide-ligands and related enzymes that are e.g. present in the bloodstream, in the extracellular matrix or on the cell membrane controlling the action of these ligands.
It is well documented that the proteolytic action of exopeptidases is one of the major degradation pathway for peptides. In order to escape attack of exopeptidases, chemical modifications of peptide termini have often been attempted. This approach is relatively simple, widely pursued and usually leads to more stable peptides of preserved biological activity. Consequently, N-terminal protection against aminopeptidases, such as acetylation or methylation, has been commonly applied to prolong the biological half-life of many peptide ligands.
It is interesting to note, that most peptide ligands conjugated to diagnostic or therapeutic moieties, such as (radio)metallated peptide ligands designed for molecular imaging or targeted therapy applications, are most often modified at the N-terminus. In general, a bifunctional chelator is covalently coupled via a carboxy functionality to the N-terminal amine of the peptide-ligand under formation of a peptide bond. While the original objective of this approach had been to introduce the metal chelate (or another medically relevant moiety), at a position as remote as possible from the receptor-recognition site to avoid interference during binding, it has inadvertently led to N-terminus capping. It is reasonable to assume that such radiometallated (or similarly conjugated) peptide ligands will therefore follow a different metabolic route than their free N-terminus counterparts and will be accordingly processed by enzymes other than aminopeptidases.
In view of the above, analogs of native receptor ligands, such as peptide-conjugates, in particular radiolabeled peptides, are expected to show sub-optimal targeting if not sufficiently modified to endure rapid enzymatic attack in the biological milieu. And in fact, this is most often observed during evaluation of many new (radio)peptide analogs. By studying the ex vivo blood of mice after administration of many radiometallated peptide ligands comprising somatostatin, gastrin, neurotensin and bombesin-like derivatives by HPLC the inventors have observed that most of these analogs were degraded to a certain extent within 5 min in vivo despite the metal-chelate coupled at their N-terminus. This finding is consistent with the inventors' assumption that proteolytic enzyme(s) other than aminopeptidases are involved in the rapid in vivo degradation of these classes of such radiolabeled peptide-conjugates.
In order to overcome problems imposed by the insufficient metabolic stability of peptide-conjugates, e.g. radiolabeled peptides, such as sub-optimal targeting and poor pharmacokinetics, painstaking research and expensive resources have been invested worldwide for the development of stabilized peptide-vectors. However, modifications undertaken to metabolically stabilize native lead-structures have often led to bioconjugates of poor interaction capacity to their cognate receptors and/or to compounds of undesirable pharmacological profile and/or sub-optimal pharmacokinetics.
It is therefore the object of the present invention to provide the means to enhance delivery of diagnostic and therapeutic agents, in particular of non- or partially stabilized peptide conjugates, optionally radiolabeled, to disease-sites.